A new strain of Aspergillus niger and its applications

Abstract

The present invention discloses a new strain of Nigrospora sphaerica and its application. A new strain of Nigrospora sphaerica, with the preservation number of CGMCC No. 41569, was preserved in the General Microbiology Center of the China Center for Type Culture Collection on September 26, 2024. The Nigrospora sphaerica is a microbial agent of strain W18. The Nigrospora sphaerica is applied in the preparation of a plant growth promoter. The application of the Nigrospora sphaerica to plants includes the following steps: S1. Preparation of spore suspension: Inoculate the strain on a PDA medium, culture until full sporulation occurs, scrape the spores, and prepare a spore suspension using a conditioning solution; S2. Seedling raising: Soak the plant seeds in sterile deionized water for one day in advance, and then soak them in sterile water at 50 - 70 °C for 1.5 - 3 h; S3. Transplanting seedlings. This application can significantly increase the relative water content, chlorophyll content, and auxin content of plant seedling leaves, has a growth-promoting effect on plants, and is a strain with excellent traits and broad application value in agriculture.

Description

Technical Field

[0001] The present invention belongs to the field of microbial technology, and particularly relates to a new strain of Nigrospora and its application. Background Art

[0002] Hazelnut, as one of the world's four well-known nuts, contains extremely rich nutritional elements, such as high content of protein, vitamin E, folic acid, eight essential amino acids for the human body, and resveratrol. Hazelnut trees belong to the genus Corylus in the family Betulaceae in terms of plant classification, and there are approximately 20 species globally. Wild hazelnuts are mainly distributed in temperate regions of Asia, Europe, and North America, and countries such as Turkey, Italy, Spain, the United States, and Portugal are the main producers. Although hazelnut trees have strong environmental adaptability and can grow under different climate conditions, they prefer cool climates and have certain requirements for specific temperature and humidity conditions. However, with the trend of global warming, the growth environment of some hazelnut trees has been affected, thus having a significant impact on their economic yield and fruit quality.

[0003] The genus Nigrospora, as a member of the Ascomycotina, Sordariomycetes, Xylariales, and Apiosporaceae, is a fungus with a wide host range and plays a key role in various ecosystems. In particular, when the genus Nigrospora is an endophytic fungus of plants, it shows significant effects in promoting host growth, enhancing development, and improving biotic and abiotic stress resistance. Therefore, the exogenous addition of Nigrospora agents has become an effective strategy to improve the adaptability of hazelnut trees, promote their growth and development, and improve the quality of hazelnuts.

[0004] Therefore, the screening and research and development of strains have continuous research value. The discovery of beneficial strains has a positive effect on enhancing the adaptability and resistance of host plants, optimizing soil quality, improving soil fertility, and promoting yield increase and quality improvement. This application focuses on screening multifunctional microorganisms that have a significant promoting effect on the growth of hazelnut trees, and the screening of this strain is of great significance for ensuring the normal growth of hazelnut trees, increasing yield, and optimizing quality. Summary of the Invention

[0005] The purpose of the present invention is to provide a new strain of Nigrospora and its application, which has a good growth-promoting effect, can improve the photosynthesis efficiency of plants, and at the same time, it can also stimulate plants to produce more auxin, thereby promoting the growth and development of plants.

[0006] In order to achieve the above purpose, the technical solution provided by a specific embodiment of the present invention is as follows:

[0007] A new strain of Nigrospora sphaerica, with the preservation number of CGMCC No. 41569, was preserved in the China General Microbiological Culture Collection Center on September 26, 2024.

[0008] In one or more embodiments of the present invention, the Nigrospora sphaerica is a bacterial agent of strain W18.

[0009] In one or more embodiments of the present invention, the Nigrospora sphaerica is applied in the preparation of a plant growth promoter. The application of the Nigrospora sphaerica on plants includes the following steps: preparing the spores of strain W18 into a suspension, and then performing root irrigation treatment on the cultivated plants with the suspension. Other management methods can adopt conventional management methods.

[0010] S1. Preparation of spore suspension: Inoculate the strain on a PDA medium, culture until full sporulation, scrape the spores, and prepare a spore suspension using a conditioning solution.

[0011] S2. Seedling raising: Soak the plant seeds in sterile deionized water for one day in advance, then soak them in sterile water at 50 - 70 °C for 1.5 - 3 h, soak them in 75% alcohol for 20 - 40 min, rinse them with sterile water 3 - 5 times, soak them in 2% sodium hypochlorite solution for 15 - 30 min, rinse them with sterile water 3 - 8 times, and evenly spread the disinfected seeds on sterilized sand for germination for 20 - 30 days.

[0012] S3. Transplanting: Sterilize peat soil, vermiculite, perlite, and sand, mix them evenly in proportion, place them in a plastic pot, transplant the seedlings into the pot containing the mixed soil, and perform root irrigation treatment with the Nigrospora sphaerica spore suspension 6 - 12 days after acclimatization. Each seedling root is inoculated with the Nigrospora sphaerica W18 spore suspension.

[0013] In one or more embodiments of the present invention, the plant is hazelnut.

[0014] In one or more embodiments of the present invention, the plant is a pear tree.

[0015] In one or more embodiments of the present invention, the spore concentration of the suspension is 8 * 106 cfu / ml.

[0016] In one or more embodiments of the present invention, the temperature for culturing and sporulating in step S1 is 20 - 36 °C, and the culture time is 5 - 10 days.

[0017] In one or more embodiments of the present invention, the conditioning solution is sterile water.

[0018] In one or more embodiments of the present invention, the mixing ratio of peat soil, vermiculite, perlite, and sand in step S3 is 6:3:3:1.

[0019] In one or more embodiments of the present invention, 10 ml of the spore suspension of Nigrospora sp. W18 is inoculated at the root of each seedling in step S3.

[0020] Compared with the prior art, a new strain of Nigrospora sp. and its application in the present invention have the following advantages;

[0021] 1) It has the effects of increasing the relative water content, chlorophyll content and auxin content of the leaves of hazelnut seedlings;

[0022] 2) The W18 strain has good adaptability, which is convenient for laboratory research and large-scale cultivation, and has a good growth-promoting effect on plants, and has potential application value as a biological fertilizer. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments recorded in the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0024] Figure 1 It is a morphological diagram of the growth of strain W18 on PDA medium and the microscopic morphological diagrams of spores and conidia;

[0025] Figure 2 It is a phylogenetic tree diagram of strain W18;

[0026] Figure 3 It is a diagram of the relative water content of leaves under the treatment of strain W18;

[0027] Figure 4 It is a diagram of the chlorophyll content under the treatment of strain W18;

[0028] Figure 5 It is a diagram of the auxin content under the treatment of strain W18. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0029] In order to enable those skilled in the art to better understand the technical solutions in the present invention, the following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0030] Example 1

[0031] As Figure 1As shown, a new strain of Nigrospora sphaerica and its application in an embodiment of the present invention. Among them, a new strain of Nigrospora sphaerica, with the preservation number of CGMCC No. 41569, was preserved in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on September 26, 2024. The address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The confirmed preserved biological name is Nigrospora coryli.

[0032] The microbial agent of Nigrospora sphaerica is the W18 strain.

[0033] Nigrospora sphaerica is applied in the preparation of a plant growth promoter. The application of Nigrospora sphaerica on plants includes the following steps;

[0034] S1. Preparation of spore suspension: Inoculate the strain TB2 on the PDA medium and culture until full sporulation. Scrape the spores and prepare a spore suspension using a conditioning solution.

[0035] Among them, the spore concentration of the suspension is 8×10⁶ cfu / ml. In step S1, the temperature for culturing and sporulating is 20 - 36°C, and the culture time is 5 - 10 days. The conditioning solution is sterile water.

[0036] Preferably, the temperature for culturing and sporulating is 28°C, and the culture is carried out for 7 days until full sporulation.

[0037] S2. Seedling raising: Soak the plant seeds in sterile deionized water for one day in advance, then soak them in sterile water at 50 - 70°C for 1.5 - 3 h, soak them in 75% alcohol for 20 - 40 min, rinse them with sterile water 3 - 5 times, soak them in 2% sodium hypochlorite solution for 15 - 30 min, rinse them with sterile water 3 - 8 times, and evenly spread the disinfected seeds on the sterilized sand for germination for 20 - 30 days;

[0038] Preferably, soak them in sterile water at 60°C for 2 h, soak them in 75% alcohol for 30 min, rinse them with sterile water 3 - 5 times, soak them in 2% sodium hypochlorite solution for 20 min, and rinse them with sterile water 5 times. Evenly spread the disinfected hazelnut tree seeds on the sterilized sand for germination for 20 - 30 days.

[0039] S3. Seedling transplantation: Sterilize peat soil, vermiculite, perlite, and sand and mix them evenly in a ratio of 6:3:3:1 and place them in a plastic pot. Select hazelnut tree seedlings with consistent growth and transplant them into the pot containing the mixed soil. After 6 - 12 days of acclimation, perform root irrigation treatment with the Nigrospora sphaerica spore suspension. Each pear seedling root is inoculated with 10 ml of the Nigrospora sphaerica W18 spore suspension (inoculation concentration is 8×10 6 cfu / ml), and set the non-inoculated treatment as the blank control CK. Harvest after 2 months of cultivation.

[0040] S4. Seedling harvesting: After 2 months of cultivation, measure the physiological indexes such as leaf water content, chlorophyll, and auxin content of the hazelnut seedlings.

[0041] Among them, the best treatment is to irrigate the roots with a Nigrospora spore suspension 10 days after the seedlings are established.

[0042] Preferably, the plant is one of Corylus heterophylla or Pyrus betulifolia Bunge.

[0043] After experiments, the results are as follows: Figure 3 As shown, it can be seen that inoculating the strain W18 significantly increases the water content in the leaves of Corylus heterophylla seedlings, thereby improving the drought resistance of the seedlings.

[0044] As Figure 4 shown, it can be seen that inoculating the strain W18 significantly increases the contents of chlorophyll a and chlorophyll b in the leaves of Corylus heterophylla seedlings, enhancing the light energy utilization rate and the rate of organic matter synthesis of the seedlings, and providing strong support for the growth and development of the seedlings.

[0045] As Figure 5 shown, it can be seen that inoculating the strain W18 significantly increases the auxin content in the roots, stems and leaves of Corylus heterophylla seedlings, which can promote the growth and development of the seedlings from multiple aspects, and provides strong technical support for the high-yield and high-quality cultivation of Corylus heterophylla.

[0046] Example 2

[0047] Isolation, screening and identification of Nigrospora sp. W18

[0048] S11. Isolation of the strain: Collect healthy and vigorous stem samples of Corylus heterophylla from the Mycorrhizal Seedling Cultivation Center (Guizhou Institute of Biology). The collected fragments were disinfected.

[0049] The specific disinfection steps are as follows: Treat with 75% ethanol for 30 seconds, rinse with sterile water, treat with 5% sodium hypochlorite for 30 seconds, rinse again, and treat with 75% ethanol again for 30 seconds, followed by three rinses. Then, dry the fragments with sterile paper and inoculate them onto potato dextrose agar (PDA).

[0050] S12. Morphological identification: After growing for 3 days, collect a hyphal tip and transfer it to PDA, and culture it at 25 °C in the dark for 7 days. Observe the growth and development with a stereomicroscope and a microscope. The results are as Figure 1 shown. The colony is flat, cottony, loose on the PDA medium, initially white, and later becomes grayish-brown due to a large amount of sporulation. The mycelium is transparent to light brown, smooth, septate, and branched. The conidiophores are similar to the vegetative mycelium, short and sparse. The conidiogenous cells are spherical or subspherical, light brown, smooth, single-budded, and determinate. The spores are spherical, subspherical or elliptical, non-budded, smooth, shiny, non-septate, and black.

[0051] S13. Molecular identification: Based on the alignment of ITS sequences and the phylogenetic tree analysis of the gene fragments of TUB2 and TEF1-α, the obtained results are as Figure 2 shown. It can be seen from the figure that strain W18 forms a unique lineage in the combined gene phylogenetic tree, is closely related to N. chinensis, and has good support. Based on comprehensive morphological identification, this strain is considered to be the new strain Nigrospora sp.

[0052] The strain W18 self-screened in this application was isolated from plant seedlings. Through morphological and molecular biological identification, this strain is the new strain Nigrospora sp. W18. By treating plant seedlings with the microbial inoculum of the new strain Nigrospora sp. W18, the relative water content of hazelnut seedling leaves increased by 18.03%, the contents of chlorophyll a and chlorophyll b increased by 5.65% and 10.95% respectively, and the auxin contents in the roots, stems and leaves increased by 140.19%, 20.37% and 145.55% respectively, indicating that the new strain Nigrospora sp. W18 provided by the present invention can significantly increase the relative water content, chlorophyll content and auxin content of plant seedling leaves, has a growth-promoting effect on plants, is a strain with excellent traits, and has broad application value in agriculture.

[0053] In summary, the strain W18 screened in the present invention has good growth performance on PDA medium, has good adaptability, is convenient for laboratory research and large-scale cultivation. After inoculating hazelnut seedlings, it is found that this strain also has a good growth-promoting effect on plants, can improve the photosynthesis efficiency of plants, and enables plants to better absorb and utilize light energy by increasing the chlorophyll content and the relative water content of leaves; at the same time, it can also stimulate plants to produce more auxin, thereby promoting the growth and development of plants. This shows that the W18 strain of this application is a strain with excellent traits, has broad application prospects and potential utilization value, and is expected to make greater contributions to agricultural production and ecological environment protection.

[0054] For those skilled in the art, it is obvious that the present invention is not limited to the details of the above exemplary embodiments, and can be implemented in other specific forms without departing from the spirit or basic characteristics of the present invention. Therefore, from any point of view, the embodiments should be regarded as exemplary and non-limiting. The scope of the present invention is defined by the appended claims rather than the above description. Therefore, all changes falling within the meaning and scope of the equivalent elements of the claims are intended to be included in the present invention. Any reference signs in the claims should not be regarded as limiting the claimed rights.

[0055] In addition, it should be understood that although this specification is described in terms of embodiments, not every embodiment contains only an independent technical solution. This narrative manner of the specification is only for clarity. Those skilled in the art should regard the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A new strain of Black Sporophycetes W18, characterized in that Its deposit number is CGMCC No.41569.

2. An application of a new strain of Black Sporophycetes W18 as claimed in claim 1, characterized in that: The black spore mold is used in preparing a hazel growth promoter, and the application of the black spore mold on hazel comprises the following steps: S1. Preparation of spore suspension: inoculate the strain on PDA medium, culture until complete spore production, scrape spores, and use a conditioning solution to prepare a spore suspension; S2. Seedling cultivation: Soak hazel seeds in sterile deionized water for one day in advance, then soak in 50-70℃ sterile water for 1.5-3h, soak in 75% alcohol for 20-40min, rinse with sterile water 3-5 times, soak in 2% sodium hypochlorite solution for 15-30min, rinse with sterile water 3-8 times, and evenly spread the sterilized seeds on sterilized sand for germination for 20-30 days; S3. Transplanting seedlings: Sterilize peat soil, vermiculite, perlite and sand, mix them evenly in proportion and place them in plastic pots. Transplant the seedlings into the pots containing the mixed soil. After 6-12 days of acclimatization, irrigate the roots with a spore suspension of Black Sporoderm. Inoculate the roots of each seedling with a spore suspension of Black Sporoderm W18.

3. The use of a new strain of Black Sporophycetes W18 according to claim 2, characterized in that: The spore concentration of the suspension is 8*10 6 cfu / ml.

4. The use of a new strain of Black Sporophycetes W18 according to claim 3, characterized in that: The spore production temperature in step S1 is 20-36° C. and the spore production time is 5-10 days.

5. The use of a new strain of Black Sporophycetes W18 according to claim 4, characterized in that: The conditioning solution is sterile water.

6. The use of a new strain of Black Sporophycetes W18 according to claim 4 or 5, characterized in that: The mixing ratio of peat soil, vermiculite, perlite and sand in step S3 is 6:3:3:

1.

7. The use of a new strain of Black Sporophycetes W18 according to claim 6, characterized in that: In the step S3, 10 ml of the spore suspension of Nigrosporium W18 was inoculated at the root of each seedling.

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